Figure 4: Regulation of p27 mRNA expression by Nczf.

(A) Nczf, p53, p21, p27, p57, p16, p19, cyclin D1, cyclin D2, cyclin D3, cyclin E1, and cyclin E2 mRNA expression in Nczf knockdown MEFs. MEFs were transfected with control or two independent siRNAs. Target-specific mRNA levels were assessed by real time PCR and normalized to Gapdh mRNA (n = 5–8). The expression in wild type was set to 1 and the relative expression of the genes of interest in Nczf−/− MEFs was calculated. (B) Expression of p27 protein in MEFs after Nczf knock down. MEFs from two independent experiments in each siRNA transfection were examined. Tubulin is shown as a loading control. Whole image of the blot is shown in supplementary information. (C) A schematic representation of Nczf binding sequence in a 5′ flanking region of p27 gene. Nucleotides that match the consensus sequence were underlined. There are multiple potential Nczf binding sites, one of them is 100% identical to the Nczf consensus binding motif (−3328), four of them are 70% compatible with Nczf consensus binding motif (−5222, −4320, −2160, and −260). (D) (left panel): A schematic representation of a 5.5 kb 5′ flanking region upstream of the translation start site of the p27 gene and reporter gene constructs. (Right panel): p27 luciferase reporter assay. MEFs were transfected with intact (p27-Luc) or Nczf binding site mutated p27 reporter constructs (p27(del)-Luc) along with Nczf knockdown vector. The cells were subjected to firefly-luciferase reporter assays with Renilla luciferase. All data of the luciferase reporter assay above are means of three independent experiments (n = 7). (E) Binding of acetyl-Histone H3 and mono/di/trimethyl-Histone H3K4 to the p27 promoter region after Nczf knockdown by 2 independent siRNAs (siRNA1 and siRNA2: closed and shaded bars, respectively) were analyzed by ChIP assay. Quantitative PCR was performed on chromatin fragments isolated before and after immunoprecipitation using anti-acetyl-Histone H3 and anti-mono/di/trimethyl-histone H3K4. Numbers in the schematic representation show targeted regions. The region −1223 fragment was used for control. (F) A schematic summary of Nczf mediated transcriptional repression of p27 gene. Abbreviations; HDAC: histone deacetylase.